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Our previous studies showed that the concentration of the β subunit of nerve growth factor (β-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β-NGF levels disappeared. Average levels of β-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions. The concentrations of mRNA for β-NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain-derived neurotrophic factor and neurotrophin-3 between the two sexes. Thus, in some regions of the brain, the production of β-NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β-NGF in these region.

Long-term diabetic patients exhibit major clinical gastrointestinal problems, such as diarrhea and constipation. In recent years, water channel protein, aquaporin 1 (AQP1) has been identified in the enteric nervous system (ENS). We have examined the pathological changes in AQP1 immunoreactive (IR) neurons in streptozotocin-induced (STZ) diabetic rats. Eight-week-old Wistar rats were injected with streptozotocin, and artificial diabetes was induced. Sixteen-week-old STZ rats were then examined with double immunofluorescence staining and ABC immunohistochemical staining. AQP1-IR neurons in STZ rats were significantly increased compared with control rats (p

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.

Most neurons in the central nervous system and peripheral nervous system do not express water transporting protein, aquaporin (AQP). In the present study, we have demonstrated the presence of AQP1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ENS) of the rat ileum. AQP1-immunoreactive (IR) neurons simultaneously expressed a neuronal marker HuC/D. Moderate numbers of AQP1-IR neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. AQP1-IR neurons can be classified as Dogiel type I cells, which have several short processes and a single long process. Many AQP1-IR fibers were found both in the myenteric and submucosal plexi. Many AQP1-IR varicose fibers were closely associated with neuronal somata in the ganglia, whereas other AQP1-IR fibers penetrated into the muscle layers. These results suggest that AQP1-IR neurons probably play a significant role within the ENS to control gut functions.

We examined whether there are the neurotransmitter candidate amino acid L-proline containing neurons localized in the rat brain. Antibodies against L-proline conjugated with rabbit serum albumin were raised in a rabbit and purified with affinity chromatography. Strong L-proline-like immunoreactivity was confined to several groups of neurons in the arcuate nucleus (n) and supraoptic n in the hypothalamus and area postrema. The brainstem had markedly stained fibers in the medial longitudinal fasciculus and localized neuronal cell body labeling in the red n, mesencephalic trigeminal n, lateral reticular n, raphe obscurus n, solitary n, compact ambiguus n, motor trigeminal n, and n of trapezoid body. Our findings are consistent with the hypothesis that L-proline may function as a neurotransmitter or neuromodulator in the brain.

To examine the immunohistochemical localization of l-arginine and l-citrulline and determine where and how nitric oxide (NO) is produced in the normal and streptozotocin (STZ)-induced diabetic rat retinas. NO is produced when l-arginine is changed to l-citrulline by NO synthase (NOS). In normal and STZ-induced diabetic rats, using an immunohistochemical method, we examined the retinal distribution of l-arginine and l-citrulline after intracardiac perfusion. We studied the distribution of NOS after immersed fixation and analyzed the number of neuronal NOS (nNOS)-positive neurons. We observed l-arginine localization in the internal limiting membrane (ILM), the ganglion cell layer (GCL), and the inner nuclear layer (INL). l-Arginine immunoreactivity in the diabetic rat retinas was found in the inner plexiform layer (IPL), as well as in the normal retina. l-Citrulline immunoreactivity in the normal and diabetic retinas was observed in the ILM, the GCL, the IPL, and the INL. nNOS staining in the normal and diabetic rat retinas was observed in the GCL, the IPL and the INL. The number of nNOS-positive amacrine cells was less in the diabetic rat retinas. NO might be produced in the GCL and amacrine cells, which show immunoreactivity to l-arginine, l-citrulline, and nNOS. In the early stage of diabetic retinopathy in STZ rat retinas, diabetes disturbed the function of the nNOS-positive amacrine cells and reduced NO production via nNOS. Jpn J Ophthalmol 2005;49:363–370 © Japanese Ophthalmological Society 2005

Background/Aims Nucleic acid damage by reactive nitrogen and oxygen species may contribute to inflammation-related carcinogenesis. To investigate the extent of nucleic acid damage in hepatitis C virus infection and its change after interferon treatment, we measured 8-nitroguanine and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the liver of patients with chronic hepatitis C (CHC) before and after interferon therapy. Methods Hepatic accumulation of 8-nitroguanine and 8-OHdG was immunohistochemically evaluated in 20 CHC patients and 7 control patients with non-alcoholic fatty liver. Results Immunoreactivities of 8-nitroguanine and 8-OHdG were strongly detected in the liver from patients with CHC, but not in control livers. 8-Nitroguanine accumulation was found not only in infiltrating inflammatory cells, but also hepatocytes particularly in the periportal area. The accumulation of 8-nitroguanine and 8-OHdG increased with inflammatory grade (8-nitroguanine; P =0.0019, 8-OHdG; P =0.0009). In the sustained virological responder group after interferon therapy, 8-nitroguanine and 8-OHdG accumulation were markedly decreased in the liver (8-nitroguanine; P =0.018, 8-OHdG; P =0.018). Conclusions In this study, we demonstrated for the first time that 8-nitroguanine accumulated in the liver of patients with CHC. 8-Nitroguanine is a useful biomarker to evaluate the severity of HCV-induced chronic inflammation in relation to hepatocellular carcinoma.

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.

Carbon monoxide (CO), an activator of soluble guanylate cyclase (SGC) and generated enzymatically by heme oxygenases (HO), is considered to function as an intra- and intercellular neuromodulator or neurotransmitter in the central and peripheral nervous systems. HO-2 is the constitutive isoform of HO and is more prevalent in nervous tissues than in the other peripheral tissues. Because previous studies have demonstrated different distributions of HO-2 in the retina depending on the species of animals, the aim of this study was to identify which cell types of the monkey retina express HO-2. The expression of HO-2 protein was examined in monkey retina by Western blot analysis. Immunoblottings from monkey homogenates revealed a single clear protein band with a molecular mass of 36 kDa that is corresponding to rat HO-2. Immunoreactivity of HO-2 was found in the perikarya of ganglion cells. Density of immunoreactive ganglion cells was higher in the central area of retina than in the peripheral retina, and somata of larger ganglion cells were stained more densely than smaller ones. In electron microscopy, immunoreactivity of HO-2 was localized on the membrane of the endoplasmic reticulum and the nuclear outer membrane of the ganglion cells. By contrast, inner plexiform layer, inner nuclear layer and outer nuclear layer were devoid of HO-2 immunoreactivity. cGMP were strongly localized in all of ganglion cells. Some cells contributed to the relatively faint cGMP staining were seen in the inner nuclear layer. In combination of HO-2 and cGMP immunocytochemistry, the overlap of co-localization of HO-2 and cGMP would suggest that HO-2 in the ganglion cells would serve as a source for CO generation and CO could serve as a gaseous signaling molecule modulator of neural activity in the retina of monkey.

Helicobacter pylori infection causes chronic inflammation, which can lead to gastric carcinoma. A double immunofluorescence labeling study demonstrated that the level of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) apparent in gastric gland epithelium was significantly higher in gastritis patients with H. pylori infection than in those without infection. A significant accumulation of proliferating cell nuclear antigen, a prognostic factor for gastric cancer, was observed in gastric gland epithelial cells in patients with H. pylori infection as compared to those without infection, and its accumulation was closely correlated with the formation of 8-nitroguanine and 8-oxodG. These results suggest that nitrosative and oxidative DNA damage in gastric epithelial cells and their proliferation by H. pylori infection may lead to gastric carcinoma. 8-Nitroguanine could be not only a promising biomarker for inflammation but also a useful indicator of the risk of gastric cancer development in response to chronic H. pylori infection.

The Goto-Kakizaki (GK) rat is a spontaneous model of non-insulin-dependent diabetes mellitus without obesity and diabetic retinopathy. We examined the retinal distribution of L-glutamate, gamma aminobutyric acid (GABA), glycine, and L-aspartate as neurotransmitters in the GK rat retina, using an immunohistochemical method with high-affinity antibodies. The retinal structures in the GK rats were the same as the controls. However, in the GK rats, immunoreactivity of L-glutamate and GABA was observed in the Müller and photoreceptor cells in addition to the immunoreactivity in normal rats. There was no change in glycine distribution between GK rats and controls. In the GK rats, L-aspartate accumulated in the inner segment of the photoreceptor cells in addition to the normal distribution. We consider that these immunoreactivity patterns in the GK rat retina might be induced by ischemia associated with diabetes mellitus.

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA2 isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.

The congenital aganglionosis rat is considered to be an animal model of Hirschsprung's disease. The mutants had a long constricted segment (from distal ileum to rectum) below the dilated distal ileum. In the dilated region, synaptophysin-immunoreactivity (IR) was almost preserved in all layers of the intestinal wall. In the constricted distal ileum and oral proximal colon, synaptophysin-IR was scarce in all layers, including the circular and longitudinal muscle layers. In the anal proximal and distal colon, synaptophysin-IR was almost scarce in the circular muscle layer (CML). An ultrastructural study confirmed that almost no terminals were found in the CML of any regions of constricted intestine. Therefore, the CML in any region of a constricted segment, is presumed to be poor innervation. However, a few synaptophysin-IR were found in the longitudinal muscle layer (LML) of an anal part of a constricted segment. An ultrastructural study also confirmed that some terminals were observed in the LML of this segment. The present study suggested that denervated CML is related to the production of constricted segment, irrespective of the presence or absence of terminals in the LML.

L-Arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.

Most of the nerve growth factor (NGF) protein in the rat and mouse brain is readily extractable in the presence of guanidine hydrochloride as is the case of brain-derived neurotrophic factor. In the present study, we measured amounts of NGF that could be extracted in the presence and absence of 1 M guanidine hydrochloride from various regions of the brains of male and female mice. About 14% of the total NGF in the hippocampus from female mice at 4 months of age could be extracted without 1 M guanidine hydrochloride (designated loosely bound NGF; about 32% in the rat hippocampus) and the remainder only in its presence (designated tightly bound NGF). The molecular masses of the NGF-immunoreactive protein in both cases were ∼ 14 kDa. There were significant differences in respective concentrations of total NGF (the loosely bound plus tightly bound NGF) in the hypothalamus and hypophysis, but not in other brain regions, between male and female mice at 4 months of age. However, levels of loosely bound NGF in the cerebellum and olfactory bulb from males were significantly higher than those in the same regions from females. This difference resulted in two-fold higher ratios of the concentrations of loosely bound to total NGF in males as compared to females. On the other hand, the ratio in the hypophysis was close to unity in both sexes. The concentrations of loosely bound NGF in the hippocampus and cerebral cortex decreased slightly with age in both males and females. Levels of loosely bound NGF increased significantly from 2 to 12 months after birth in the whole brain, olfactory bulb, cerebellum, hypothalamus and hypophysis to a greater extent in males than in females. Thus, it is suggested that high ratios of loosely bound to total NGF in selected regions of brains from male mice are due to an enhanced conversion from tightly to loosely bound form, which is considered to be regulated by androgens (see Brain Res. 322, 112–117, Brain Res. 322, 1990 ). They may also influence the total NGF expression.

Age-related changes in the levels of brain-derived neurotrophic factor (BDNF) in selected regions of brains from rats, normal mice and senescence-accelerated mice were compared to those of nerve growth factor (NGF) and neurotrophin-3 (NT-3). The concentration of BDNF increased with age in the rat hippocampus while it decreased in the rat cerebral cortex. The level of BDNF in the hippocampus from aged rats was about 260%, of that in the same region from young adult rats. A strong staining with antibodies specific for BDNF was observed in the hilus of the dentate gyrus in the hippocampus from aged rats. By contrast, BDNF levels were significantly lower in four brain regions from aged rats as compared to young adult rats (30, 56, 52 and 52%, lower in the septum, cerebral cortex, cerebellum and striatum, respectively). Patterns of age-related changes in the level of BDNF in the mouse hippocampus. cerebral cortex, cerebellum and olfactory bulb were similar to those in the respective regions from rats. In rats, the concentration of NGF decreased with age in the cerebral cortex but remained unchanged in the hippocampus, cerebellum and olfactory bulb. In mice, levels of NGF increased in all four brain regions from 1 to 18 months after birth. The concentrations of NT-3 increased and decreased with age in the rat cerebral cortex and cerebellum, respectively, while minimal changes were observed in the rat hippocampus and olfactory bulb as was also true in mice. In senescence-accelerated mice with memory disturbances, no marked increases in levels of NGF and BDNF in the hippocampus and in the level of NT-3 in the cerebral cortex were found. Thus, increases in levels of BDNF and NT-3 occurred in the murine hippocampus and cerebral cortex, respectively, during normal aging, but not during aging of mice with pathological changes.

Recent studies have shown that carbon monoxide (CO) may function as a gaseous signaling molecule in a similar way to nitric oxide. In the gastrointestinal tract, immunoreactivity against a CO-producing enzyme, heme oxygenase-2 (HO-2), was reported in epithelial cells and neurons of submucosal and myenteric plexus. However, details of the epithelial cells in the gastric mucosa remain unknown. The aim of this study was to clarify if mRNA for HO-2 is expressed in the rat stomach, if HO-2 protein is present in the mucosa, and to define the cell types of the HO-2-immunoreactive cells. HO-2 mRNA and protein were detected in fundic and pyloric mucosa of rat stomach using an RNA protection assay and western blot analysis. Immunohistochemical study showed that HO-2 was localized in parietal cells of the fundic glands and gastrin cells of the pyloric glands of both rat and monkey. The results suggest that HO-2 enzyme is produced in the gastric mucosa, and that CO is released from parietal cells and gastrin cells.

Background and Purpose —Subarachnoid hemorrhage (SAH)-induced heme oxygenase-1 (HO-1) in glia throughout the rat brain without affecting heme oxygenase-2 (HO-2). However, the relationship between cerebral vasospasm and the expression of heme oxygenases after SAH is thus far unknown. The purpose of the present study was to clarify the effect of vasospasm on the expression of heme oxygenases in a rat model of SAH. Methods —Endothelin, hemolysate, hemolysate saturated with carbon monoxide (CO-hemolysate), and saline were injected into the cisterna magna of adult rats. Angiography was repeated before each injection and 15 and 60 minutes and 24 hours after each injection. Immunocytochemistry for HO-1, HO-2, and glial fibrillary acidic protein (GFAP) was performed 24 hours after the injection. Results —A significant vasospasm occurred in the basilar artery after the injection of endothelin, hemolysate, and CO-hemolysate. The degree of vasospasm was most prominent 15 minutes after each injection. There was no significant difference in the degree of vasospasm among injections. The HO-1 was induced exclusively in the glial cells throughout the brain after injection of hemolysate and CO-hemolysate; however, it was not induced by endothelin and saline. In the dentate gyrus of the hippocampus and the molecular layer of the cerebellum, the HO-1−positive cells were also stained for GFAP, suggesting astrocytic glial cells. On the other hand, HO-2 immunoreactivity was abundant in neurons and was not affected by endothelin, hemolysate, CO-hemolysate, or saline. Conclusions —It is suggested that heme per se, rather than ischemia induced by vasospasm, plays a pivotal role in the expression of HO-1 in this rat model.

Recent studies suggest that carbon monoxide (CO), which is formed by the enzyme heme oxygenase (HO) during the conversion of heme to biliverdin, shares some of the chemical and biological properties of nitric oxide (NO) and may play roles similar to those of NO. Heme oxygenase activity in the kidney has been reported for many years, and there are some reports on the expression of mRNA for two HO isozymes (HO-1 and HO-2) and cellular localization of HO-1 protein. However, cellular localization of HO-2 protein in the kidney under normal conditions has not been reported. In the present study we examined the expression and distribution of HO-2 mRNA and HO-2 protein in rat kidney using RNA protection assay and light and electron immunocytochemistry. RNA protection assay confirmed constitutive expression of HO-2 transcript in rat kidney. HO-2 immunoreactivity was selectively found in epithelial cells of the thick ascending limb and distal convoluted tubule, connecting tubule cells, and principal cells of the collecting duct. These results suggest that HO-2 is synthesized in the kidney and that HO-2 in the epithelial cells of renal tubules may serve as a source for CO generation under normal conditions.

In previous studies, the localization of a pertussis toxin-sensitive G protein was demonstrated in ependymal cilia, but the identification of the subtype of G protein was inconsistent. To clarify this issue, we studied the localization of Goalpha, Gi1alpha, Gi3alpha and Gi2alpha in the ciliated ependymal cells and in the cilia of some other tissues of rats using specific antibodies. The cilia of the ependymal cells that line the ventricular cavity of the brain were intensely immunoreactive for Gi2alpha, but not for Goalpha, Gi1alpha or Gi3alpha. Immunoblot analysis demonstrated higher levels of Gi2alpha in the ependymal cilia-rich pellet than in the motor area of the parietal cortex. At the ultrastructural level, the immunoreactivity specific for Gi2alpha was found predominantly in the cilia, but rarely in the microvilli or the basal bodies of ependymal cells. In cross-sections, the immunoreactivity specific for Gi2alpha was observed only in cell membranes, in particular, in the inner electron-dense leaflet of the trilaminar structure. In addition to that in the ependymal cilia, such specific localization of Gi2alpha was observed in the motile cilia in other tissues, including the oviduct and trachea. By contrast, the stereocilia in the ductus deferens were not immunopositive for Gi2alpha. These findings suggest that Gi2 might play an important role in the signal transduction in ciliary membrane-associated function(s) of the ependymal cells, oviduct and trachea.

We investigated immunohistochemically the distribution of γ-aminobutyric acid (GABA) and the calcium-binding protein parvalbumin (PV) in postnatal rat retina. At birth, GABA was found in the ganglion cell layer and in some cells in the nuclear layer that differentiate to amacrine cells and horizontal cells, while PV appeared in some cells in the outer portion of the nuclear layer that differentiates to horizontal cells. At P7, when the inner and outer nuclear layers were divided by outer plexiform layer, both GABA and PV were observed in some cells in the ganglion cell layer, amacrine cells, and horizontal cells. As the postnatal day advanced, GABA and PV were observed in some cells in the ganglion cell layer and amacrine cells. PV has been present in the horizontal cells after its appearance, however, the immunoreactivity of GABA was gradually weaker as the postnatal day advanced. Since GABA existed transiently in the horizontal cells in the early postnatal period, it seems that GABA may have some specific function for differentiation of horizontal cells. However, PV has existed in the horizontal cells since they differentiated in the retina, suggesting that PV may be one of the specific marker of horizontal cells.

Carbon monoxide (CO) is a gas that can permeate biological membranes and it has been suggested that the gas plays a signaling role in the brain by activating guanylyl cyclase (sGC). CO is generated by heme oxygenase during the conversion of heme to biliveridn. In this study, we raised an antiserum against the chemically synthesized amino-terminal fragment of heme oxygenase-2 (HO-2) and studied the distribution of this enzyme in the rat cerebellum by an immunocytochemical method. Immunoreactivity specific for HO-2 was observed only in neurons. In the Purkinje cells and the basket cells of the cerebellum, immunoreactivity was detected in the dendrites and the somata but not in the axon terminalis, suggesting that CO might be liberated primarily from the dendrites and somata rather than from the axons in this region of the rat brain.

Biochemical studies have revealed considerable amounts of free amino acids in the kidney. We examined the intrarenal distribution of three amino acids (aspartate, glutamate, and taurine) in the rat kidney with an immunoperoxidase method. In the renal cortex, all three amino acids were concentrated in the renal corpuscles and in the epithelia of the collecting tubules. Immunostaining of the collecting tubules was more intense in the principal cells than in the intercalated cells. The distal convoluted tubules were also immunostained with aspartate- and glutamate- specific antibodies but not with the taurine-specific antibody. In the renal medulla, the immunoreactivity specific for aspartate and for glutamate was similar; it was weak in the thick portion of the loop of Henle and strong in the collecting tubules. Immunoreactivity specific for taurine was restricted to regions within the epithelia of the thin portion of the loop of Henle and the collecting tubules. The significance of the accumulated amino acids as osmoregulatory agents is discussed.

An investigation was made of age-related changes in levels of the beta-subunit of nerve growth factor (beta-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of beta-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of beta-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of beta-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of beta-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.

d-Serine is a novel candidate for an intrinsic ligand for the glycine site of N-methyl-d-aspartate (NMDA) receptors in mammalian brain. d-Serine and serine racemase, which produces d-serine from l-serine, have long been presumed to be localized in astrocytes. However, we have reported that d-serine immunoreactivity was observed in neurons in rats. In the present study, the distributions of d-serine and serine racemase were investigated in combination with marker proteins for neurons, astrocytes and oligodendrocytes in mice. Immunoreactivities for d-serine and serine racemase were found in neurons and oligodendrocytes. These results suggest that d-serine can be produced in neurons as well as glias and used as a neurotransmitter, which control the synaptic function of NMDA receptors.

Gamma-aminobutyric acid (GABA) and glycine are known as major inhibitory neurotransmitters in the intermediolateral nucleus of the spinal cord. Distribution and density of GABA immunoreactive axon varicosities and glycine receptor immunoreactive dendrites and somata in the intermediolateral nucleus were examined by immuno-electronmicroscopy. GABA immunoreaction was observed in the axon varicosities of axo-dendritic and axo-somatic synapses. Glycine receptor immunoreaction was seen in association with the postsynaptic membrane of dendrites and soma. GABA immunoreactive axon varicosities were larger (1.01 +/- 0.49 x 1.20 +/- 0.38 microns) than axon varicosities presynaptic to glycine receptors (0.72 +/- 0.22 x 0.98 +/- 0.33 microns). The density of GABA immunoreactive axon varicosities was 3.65/100 microns 2 and that of glycine receptor immunoreactive synapses was 4.78/100 microns 2. A subpopulation of GABA immunoreactive axons (42%) made synaptic contact with glycine receptor immunoreactive dendrites or soma, indicating the coexistence of GABA and glycine.

Development of extraocular muscles of rat was studied immunohistochemically using antibodies against three molecular markers of muscle differentiation: brain type (CK-B) and muscle type (CK-M) creatine kinase isoenzymes and muscle type enolase isoenzyme (β-enolase). The time of innervation of extraocular muscles was also studied immunohistochemically using antibody against neuron specific enolase (NSE). Periodic acid-Schiff (PAS) and PAS after diastase digestion stainings was used for the demonstration of glycogen. At embryonic day (E) 15, when muscle primordium of each extraocular muscle appears, β-enolase and glycogen were observed in all muscle primordia, while CK-B was immunoreactive only in lateral rectus (LR), superior rectus (SR), inferior rectus (IR) and inferior oblique (IO) muscle primordia. CK-B immunoreactivity appeared in superior oblique (SO) and medial rectus (MR) muscles at E17 and E18, respectively. By E18-19, CK-M immunoreactivity became positive in all muscles. NSE immunoreactive nerve fibers were first observed in LR, SR, IR and IO at E15, in SO at E16, and in MR at E17. Consequently, β-enolase immunoreactivity and glycogen appeared in all extraocular muscles at E15, while CK-M at E18. LR, SR, IR and IO muscles became immunoreactive to CK-B antibody at E15; however, SO and MR muscles became immunoreactive at E17 and E18, respectively. The sequence and time of appearance of CK-B in extraocular muscles were similar to those of NSE-immunoreactive nerve fibers. These findings suggest that in rats the expression of CK-B in each extraocular muscle coincides with the innervation of that muscle, while CK-M is expressed only after innervation.

p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.

Changes in parvalbumin-like immunoreactivity were studied during ontogenetic development of the central auditory system of the Mongolian gerbil ( Meriones unguiculatus ). The nucleus of the trapezoid body contained cells that could be stained on the day of birth while in the superior olivary complex, prominent staining of cell somata was only found at P15 (postnatal day 15). Neurons located in the ventral nucleus, in the dorsal nucleus of the lateral lemniscus and in the inferior colliculus developed parvalbumin immunoreactivity mostly between P11–P15 (ventral nucleus), P15–P19 (dorsal nucleus) and P15–P19 (inferior colliculus), respectively. The accumulation of PV correlates reasonably well with the functional maturation of the neurons in these areas.

The coexistence of glycine- and PV-immunoreactivities was studied immunocytochemically in the nuclei of the superior olive, trapezoid body, cochlea and lateral lemniscus. All of the PV-immunoreactive neurons in the nuclei of the superior olive and trapezoid body were immunoreactive to glycine but not to GABA. In the dorsal cochlear nucleus, PV-positive neurons were sometimes immunoreactive to glycine. In the ventral nucleus of the lateral lemniscus, PV-positive cells were immunoreactive neither to glycine nor to GABA. Consequently, it was concluded that PV-immunoreactivity was distributed not only in the GABAergic neurons, but also in the glycinergic neurons and possibly in wider neuronal populations.

In our previous report, we have shown the sex difference in the concentration of the beta-subunit of nerve growth factor (beta-NGF) in the neural and paraneural tissues of mice. In this investigation, we examined the effects of castration of adult males, and of neonatal and/or adult treatments with testosterone on levels of beta-NGF in the several tissues of mice. Castration caused a marked reduction in the levels of serum testosterone and of beta-NGF in the brain, spinal cord and submandibular glands, but not in the pancreas and kidneys. Continuous infusion of testosterone for one week into adult males that had been castrated at 2 months of age restored the level of beta-NGF in the three tissues mentioned above. A single injection of testosterone to 5-day-old female pups to masculinize the brain gave no effect on the level of beta-NGF in any tissue dissected after 4 months. A one-week infusion of testosterone into adult females slightly increased levels of beta-NGF in the brain and spinal cord, but the same treatment of adult females given in advance a single dose of testosterone at 5 days of age caused a significant increase in its levels over those of untreated females. These results suggest that neonatal and adult exposures to testosterone can influence the endogenous concentration of beta-NGF in the brain and spinal cord.

The alpha-subunit of a GTP-binding protein Go (Go alpha), is a new marker protein of neurons and neuroendocrine cells. In our previous report, this protein was shown to be also distributed in the cardiac muscle of young rats, although the cardiac muscle is traditionally thought to be a non-neuronal tissue. To evaluate this unusual finding, expression of other neural marker proteins in the heart was examined. Immunohistochemical studies using antibodies against gamma-enolase, neurofilament and Go alpha protein revealed localization of these neural markers only in neurons and neuroendocrine cells in adult rats. In young rats, however, these neural markers were localized not only in these cells but also in the cardiac muscle. An enzyme immunoassay study of enolase isozymes showed that the heart of young rats was rich in neuron-specific gamma-enolase rather than in muscle-specific beta-enolase, while the heart of adult rats was deprived of the former and rich in the latter. These results suggest that the heart of young rats should be understood as a neuroendocrine tissue as well as a muscular tissue.

5-Bromo-2'-deoxyuridine (BrdU) and 5-chloro-2'-deoxyuridine (CldU) were sequentially administered intraperitoneally into mice at 1-hr intervals. After one additional hr, the small intestines were resected, fixed, and embedded in paraffin. In histological sections stained with monoclonal antibody Br-3 reactive to both BrdU and CldU, and CldU antibody reactive only to CldU, three types of staining could be identified in the proliferating zone. Cells with nuclei stained only with Br-3 antibody were estimated to have completed DNA replication during the first 1 hr and were fixed in G2/M-phase. Those nuclei were frequently found in apical areas of the simple columnar epithelium of the intestine, whereas other nuclei were located basally in the cells. This observation suggested intracellular movement of cell nuclei in G2/M-phase. Identification of cells in early S-phase became possible using these antibodies in combination with DAB and fluorescence stainings. Replication sites in early S-phase nuclei were found to be numerous, whereas in late S-phase they were larger in size and much smaller in number.

Increased cancer risk occurs in inflammatory bowel disease (IBD) undergoing long-term chronic inflammation. To evaluate whether inducible nitric oxide synthase (iNOS)-dependent DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to female severe combined immunodeficiency (SCID) mice. CD45RBhighCD4+ T cells were isolated from mouse spleen after staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD45RB monoclonal antibody, followed by anti-FITC-conjugated microbeads. This IBD mouse model showed that the bodyweight increased with aging to a lesser extent than non-treated controls, and that the intestine was shortened. Pathological findings of this mouse model, which showed severe inflammation in colon tissues, were similar to IBD patients. Double immunofluorescence technique revealed that both 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were formed mainly in epithelial cells of the IBD mouse model. 8-Nitroguanine was formed in most of 8-oxodG-immunoreactive nuclei of epithelial cells. iNOS, proliferating cell nuclear antigen and p53 protein were also expressed in the colon epithelium. These results indicate that nitrative DNA damage, as well as oxidative DNA damage, is induced in colon epithelial cells of the IBD mouse model followed by proliferation of these cells, which may contribute to colon carcinogenesis.

l-Aspartate (l-Asp) is an excitatory neurotransmitter in the central nervous system. In the present study, we demonstrate, for the first time, the presence of l-Asp in a particular neuronal cell class in the enteric nervous system (ENS). Scattered l-Asp-immunoreactive neuronal cell bodies and nerve fibers were found extensively in both the myenteric and submucosal plexus throughout the small and large intestines. Many l-Asp-immunoreactive nerve fibers, which originated from intrinsic nerve cell bodies, were found in the ganglia and interconnecting nerve bundles. Electron microscopy revealed that l-Asp-immunoreactive terminals frequently formed synaptic contacts with intrinsic nerve cells, suggesting that some l-Asp-immunoreactive neurons might function as interneurons. These results suggest that l-Asp-immunoreactive neurons play a significant role within the ENS to control intestinal functions. The presence of enteric l-Asp-immunoreactive neurons provides strong support for the proposal that l-Asp is a neuromodulator in the rat ENS.

Chronic inflammation induced by repeated infection with Opisthorchis viverrini has been postulated to be a risk factor for cholangiocarcinoma. To clarify the mechanism of carcinogenesis induced by repeated O.viverrini infection, we investigated the timecourse of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, inducible nitric oxide synthase (iNOS) expression, nitric oxide production and pathological features in hamsters with two (2-IF) or three (3-IF) O.viverrini infections. Inflammatory cell infiltration triggered by repeated infection (3-IF > 2-IF > 1-IF) was earlier than by single infection (1-IF). HPLC coupled with an electrochemical detector revealed that 8-oxodG level in the liver was the highest on day 3 in 3-IF and day 7 in 2-IF, earlier than that on day 21 in 1-IF. Notably, a double immunofluorescence study revealed that formation of 8-nitroguanine and 8-oxodG appeared to increase in the epithelium of bile ducts in the order 3-IF > 2-IF > 1-IF after the decrease in inflammatory cells. This may be explained by the fact that repeated infection increased iNOS expression in the epithelium of bile ducts in the order 3-IF > 2-IF > 1-IF on day 90. Proliferating cell nuclear antigen accumulated in the epithelium of bile ducts on day 90 after repeated O.viverrini infection, supporting the hypothesis that cell proliferation was promoted by inflammation-mediated DNA damage. In conclusion, more frequent O.viverrini infection can induce the expression of iNOS not only in inflammatory cells but also in the epithelium of bile ducts and subsequently cause nitrosative and oxidative damage to nucleic acids, which may participate in the initiation and/or promotion steps of cholangiocarcinoma development.

Nucleic acid damage by reactive nitrogen and oxygen species may contribute to the carcinogenesis associated with chronic infection and inflammation. We examined 8-nitroguanine and 8-oxo-7,8-dihydro-2 ′ -deoxyguanosine (8-oxodG) formation and nitric oxide (NO) production in hamsters infected with Opisthorchis viverrini (OV). Formation of 8-nitroguanine was assessed immunohistochemically with an antibody specific for 8-nitroguanine. 8-Nitroguanine formation was found mainly in the cytoplasm and slightly in the nucleus of inflammatory cells and epithelial lining of bile duct at inflammatory areas in the liver. 8-Nitroguanine immunoreactivity reached the highest intensity on day 30. A time profile of 8-nitroguanine formation was closely associated with that of plasma nitrate/nitrite. HPLC with an electrochemical detector revealed that the amount of 8-oxodG in the liver reached the maximal level on day 21. The mechanisms of 8-oxodG and 8-nitroguanine formation via O 2 − and NO production triggered by OV infection were discussed in relation to cholangiocarcinoma development.

Taurine is one of the most abundant free amino acids in animal tissues. It is not incorporated into proteins but found mainly in the free form. Taurine has long been regarded as a mere end product of sulphur amino acid metabolism with no physiological role except that of bile acid conjugation. Yet, recent studies have implicated taurine in many physiological events, including osmoregulation, anti-oxidation, detoxification, membrane stabilization and neuromodulation1–4. However, its localization and function in the stomach has not been previously analysed in detail. The aim of this study is to reveal the distribution of taurine in the rat stomach, and to define taurine-containing cells in this organ by immunohistochemical techniques.

To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca(2+)-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.

L-Serine has been suggested by in vitro studies to be an important neurotrophic factor which supports survival and neurite outgrowth of neurons. It is also a precursor of D-serine, a putative neurotransmitter. In the present study, we raised antibodies against L-serine in a rabbit and examined immunohistochemical distribution of the amino acid in the rat brain. In the hippocampus and the cerebellar cortex, where neurotrophic effects of L-serine have been indicated, L-serine immunoreactivity was found primarily in astrocytes. In the brain stem, where neuronal distribution of D-serine was reported, positive staining for L-serine was located primarily in neurons. Regional differences of cellular distribution of L-serine I were indicated.

D-Amino acids are thought not to occur in mammalian tissues. However, previous studies reported D-serine was present only in astrocytes in the rat brain. In the present study, it was indicated by a highly sensitive immunocytochemical method with a D-serine specific antibody that D-serine was contained not only in astrocytes but also in some neurons, such as pyramidal neurons in the cerebral cortex, and neurons in the nucleus of the trapezoid body. Some amacrine cells also showed strong immunoreactivity for D-serine in the eyes which were injected with colchicine into the corpus vitreum.

Levels of neurotrophin-3 markedly decrease in the rat cerebellum after the first 10 days of life, suggesting an importance during early development. To further examine the effect of neurotrophin-3 on the developing cerebellum, we injected a monoclonal antibody against neurotrophin-3 into the lateral ventricle of 7.5-day-old rats. The resultant depletion of neurotrophin-3 caused a significant decrease in cerebellar wet weights noted at 7 and 23 days thereafter. Other changes noted 48 h after injection of monoclonal antibodies against neurotrophin-3 included reduced incorporation of bromodeoxyuridine into granule neurons in the external germinal layer, an elevated density of atrophic neurons that had just migrated under the Purkinje cell layer, and an increased number of apoptotic neurons in the internal granule cell layer. These changes were limited to the central lobe. The concentration of neurotrophin-3 protein in the posterior region, including the central lobe, was about four- and threefold higher than that in the anterior region of the cerebellum of 9.5- and 30-day-old rats, respectively. Immunocytochemical examination showed higher amounts of neurotrophin-3 protein in the central lobe than in the anterior lobe. Our results provide evidence that neurotrophin-3 regulates the proliferation of granule precursors and supports the survival of mature granule neurons in restricted lobules, suggesting an involvement in limited regions at a specific stage in development of the rat cerebellum.

Recent studies have shown that free d-serine, an agonist for the N-methyl-d-aspartate receptor, is present in the forebrain of rats. In the present study, we raised antibodies against d-serine and examined the distribution of both endogenous and intracerebroventricularly administered d-serine in the rat forebrain by an immunocytochemical method. d-Serine-like immunoreactive cells were found in glial cells in the brains of the rats injected with d-serine into the lateral ventricle. No immunoreactive cells were seen in the brains of untreated rats. The results suggest that glial cells may accumulate and catabolized d-serine to regulate the concentration of this neuroactive amino acid in the forebrain.